Vibrio vulnificus is a highly virulent estuarine bacterial species that has been implicated as a cause of gastroenteritis, serious wound infections, and septicemia, with mortality rates for patients with septicemia exceeding 50%. Along the US Gulf coast infections with V. vulnificus account for over 50% of all reported Vibrio-associated illness. V. vulnificus produces an extracellular cytotoxin/cytolysin that is cytolytic for CHO cells, hemolytic, and mouse lethal (IV LD50=80ng of purified protein). It has been hypothesized that this cytolysin is a major virulence factor for the organisms; other studies, however, suggest that production of toxin is not important in actual infections. Documentation that the toxin is critical for pathogenicity will help us in our understanding of the rather striking disease process associated with V. vulnificus infections, and may form the basis for development of specific therapy for use in acute cases. A better understanding of the role of this cytotoxin in disease will also help us in defining the role played by cytotoxin/hemolysins produced by other Vibrio species, including V. cholerae, V. parahaemolyticus, V. damsela, V. fluvialis, and V. furnissii. Side benefits of this work include development of a highly specific genetic probe and an epidemiologic typing system for V. vulnificus, as well as possible development of serologic assays for infection. In this proposal we will use molecular genetic and immunologic techniques to investigate the role of the cytolysin in pathogenicity. In our laboratory we have already purified the cytolysin, and isolated the genes encoding it on a 3.2 kB DNA fragment. The subcloning and sequencing of these genes will enable us to identify the region of DNA required for cytolysin production; using these data, we will delete the gene from a fully virulent strain by a marker exchange procedure. Comparison of the virulence of these isogenic strains with and without the cytolysin genes in animal models will allow us to examine the contribution of this cytolysin to the ability of V. vulnificus to cause disease. We will use our purified cytolysin to develop ELISAs to permit rapid screening of isolates for cytolysin production, and of sera for the presence of antibodies to cytolysin. Although the proposal will focus on the role of the cytolysin in pathogenicity, if time permits we will expand the study to look at other possible virulence factors, including siderophores and iron-associated outer membrane proteins.